Dispenser system for mass spectrometric sample preparations

ABSTRACT

The invention relates to the preparation of samples on mass spectrometric sample supports with dispensing of liquids, and particularly to devices and methods to clean the dispenser. During dispensing of hundreds of samples, solved material may crystallize over time as deposit at the capillary tip of the dispenser, which impedes the vertical detachment of the drop in the medium and long run. Therefore, frequently cleaning the capillary tip is essential for a robust operation of the preparation device. The invention proposes to automatically clean the dispenser tip by creating a drop of washing fluid, such as pure solvent, fully enclosing the dispenser tip. The washing fluid is fed through a channel outside the central dispenser capillary to a location slightly above the capillary tip. The size of the hanging drop of washing fluid is photometrically regulated and monitored to prevent it from falling off prematurely.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to the preparation of samples on massspectrometric sample supports with dispensing of liquids, andparticularly to devices and methods to clean the dispenser.

Description of the Related Art

The preparation of samples for ionization by matrix-assisted laserdesorption (MALDI) or similar ionization methods requires the use oforganic solvents to apply the solution containing matrix material ontothe individual samples under analysis. These samples have to be appliedto the sample support manually or with the aid of pipetting robots. Thesolvents must subsequently be vaporized in order to allow crystals ofthe matrix substance, into which the analyte substances have to beembedded, to grow. Since ionization by matrix-assisted laser desorption(MALDI) and its requirements are widely known, no detailed descriptionwill be given here.

Today, ionization by matrix-assisted laser desorption is used widely forthe mass spectrometric identification of microbes. This identificationof microbial samples involves the daily preparation of hundreds ofthousands of samples in many hundreds of microbiological laboratories.The preparation of microbes should serve here as an example of samplepreparation.

Since only the substances from inside the microbe cells are usable forthe mass spectrometric identification, the microbe cells must first becell disrupted. This cell disruption also takes place predominantly onthe sample support. The first step is to apply small, hardly visiblequantities of around 10⁵ to 10⁷ microbes from agar plate colonies ontothe test sites of the sample support. This transfer of microbes iswidely done manually, but there are automatic devices available for thispurpose. The cells of the microbes are usually cell disrupted on thesample support by strong acids, which must subsequently be dried up byvaporization. The acids used for this purpose are 70-percent formic acid(boiling point 101° C.; vapor pressure 43 hPa at 20° C.) ortrifluoroacetic acid of similar concentration (TFA; boiling point 72°C.; vapor pressure 110 hPa at 20° C.). Quantities of around onemicroliter are applied onto each sample. When they have dried, thematrix solution is applied, also in quantities of around one microliter.The matrix solution usually contains a solid organic acid (usuallyα-cyano-4-hydroxycinnamic acid, HCCA, but also 2,5-dihydroxybenzoicacid, DHB, for example) in a solvent mixture of acetonitrile andalcohols. For reasons of occupational health and safety, ethanol isusually used, although methanol would be the better alternative. If thecell walls have not yet been completely destroyed by the acid, thematrix solution penetrates into the microbes through the weakened cellwalls and causes them to burst by osmosis. Soluble contents, inparticular the soluble proteins, then dissolve in the matrix solution.The drying of the matrix solution causes tiny crystals of matrixsubstance to form, into whose crystal lattice or crystal boundariesmolecules of the contents are embedded. The microbes are then identifiedwith the aid of a mass spectrum of the contents.

The sample supports are usually the size of microtitration plates (or afraction thereof) and nowadays usually have 48, 96 or 384 visible testsites for the application and preparation of the samples. Samplesupports with 1536 test sites are also in use. The test sites withdiameters of 0.8 to 3.0 millimeters can be identified in someembodiments with the aid of milled-in rings, whose sharp milled edgesprevent the applied acids and solvents from flowing laterally away. Thetest sites can also take the form of hydrophilic areas in a hydrophobicenvironment.

The sample support can especially also contain small pins, around 2millimeters in diameter, which are inset into the sample support so asto be flush with the surface. The pins can be individually loaded withmicrobes by direct contact with microcolonies on agar surfaces. This cangreatly shorten the culturing times, as is disclosed in the patentapplication WO 2013/182648 A1 assigned to the Applicant. The holes forthe pins can have a slight chamfer, which keeps the edges of the pinsclear so that the surface tension of the liquid applied to the end ofthe pins prevents it from running over the edge of the pins.

At present, the preparation is largely carried out manually withdispensing pipettes, without a hood, because hoods are rare inmicrobiological laboratories. This can be a health hazard if theventilation is insufficient. Even when a hood is available, it is oftennot used because accurate pipetting onto a small sample spot in an openhood is very awkward. So, devices for automatic preparation whichautomate the application of the acids and the matrix solutions, andpreferably do not release hazardous vapors so that a hood is notrequired, are desirable.

Liquids in quantities of around one microliter do not drip by their ownweight even from very fine pipette tips, but are applied according tothe prior art by dabbing them onto the sample. A new pipette tip must beused for each sample in order to prevent samples being transferred.Non-contact application of the liquids onto the samples is particularlyadvantageous because it eliminates the need to replace the pipette tipseach time.

The generation of free-flying droplets with volumes of only a fewnanoliters is known particularly from printing technology; some systemsoperate with piezo technology, others with vapor-bubble technology. Withthis technology, the droplets are ejected from nozzles. This technologydoes not lend itself to the application described here because almost athousand tiny droplets, whose surface area is large relative to theirvolume, would have to be applied to a test site, preferably without anyevaporation at all. Since matrix solutions near the saturation limithave to be used, there is a risk that deposits will form on the nozzleoutlets at an early stage, meaning that the drops no longer leave thenozzle in the correct direction, and that the matrix solution willcrystallize out prematurely in the flying droplets.

Dispensers which apply liquids with volumes of around one microliteronto test sites positioned below the dispenser, and which operate withfalling drops of correct size without direct contact of the dispensertip to the sample site, are also known in principle and can be usedhere. For this type of non-contact application, it is necessary toposition the dispenser exactly vertically above the test site (or thetest site on the sample support vertically below the dispenser). For thedispensing of these small quantities of liquid onto the test sites,there are different technical solutions, such as detaching the drop bypressure surges in the liquid feed, acoustic shock waves generated bypiezo crystals, or sudden vertical movement of the capillary tip.Particularly simple and low-cost is a dispenser unit which has outer gaschannels, arranged symmetrically around the central capillary andpointing to the tip of the central capillary. There may be a singleannular channel by a concentric capillary around the dispensingcapillary, or there may be an arrangement of at least two, preferablythree or four single channels. A tiny pump presses a drop of around onemicroliter out of the central capillary; a small pressure surge of airor other suitable gas through the surrounding gas channel(s) then stripsthe hanging drop of dispensing liquid from the central capillary andcauses it to fall vertically onto the test site. A simple arrangement isshown in FIG. 1, showing two concentric capillaries, the inner for thedispensing liquid, the outer for the gas surge. The pressure surge ofthe gas, the height of the fall and the fall speed of the droplet mustbe small enough so that the drop does not splatter, but large enough sothat the drop does not simply roll away on the sample surface. Thenon-contact deposition of the liquid drops means that replaceablepipette tips are no longer required for the preparation.

Because quite often matrix solutions near saturation have to be used toprepare the samples for a mass spectrometric analysis, the function ofthe dispenser is repeatedly disturbed by crystals of matrix materialformed and growing at the tip of the dispenser over time. Quite oftenwet crystals form at the outside (or outer circumference) of the tip,creeping slowly upwards at one side of the tip during subsequentdispensing cycles. When such obstruction exists, there is a danger thatthe drop of dispensing liquid, prior to being detached, is drawn upwardsat one side of the tip by the easy wettability of the deposit incontrast to the commonly low wettability of the tip material. As aconsequence, the drop stripped off by the gas surge will not fall justvertically and eventually could miss the sample site on the samplesupport. According to the prior art, the dispenser has to be dismountedand cleaned frequently; for that purpose, at least the inner capillaryhas to be emptied, then filled with washing fluid (in most cases a pureliquid solvent). The tip has to be washed by rinsing inside and outside.Then the washing fluid is removed from the tip, and dispensing fluid hasto be refilled.

Dispensers can be equipped with drop size regulation systems, likewiseknown since long (see, for instance, publication JP 1986-231461; Apr. 5,1985; F. Sugaya, describing a photometric drop size measuring andregulating system; also U.S. Pat. No. 5,601,980 A or US 2006/0144331A1).

The present disclosure references ionization by matrix-assisted laserdesorption (MALDI), where ions are produced during the desorption bypulsed laser beams. It goes without saying that sample preparations forother types of ionization shall also be possible where, for example, theanalyte substances in the prepared samples are first transferred intothe gaseous phase, and only then ionized. Simple laser desorption incombination with chemical ionization (LDCI) can be carried out, forexample, as can direct electrospray ionization from the surface (DESI),but other types of ionization can also be used. Accordingly, the term“ionization with matrix-assisted laser desorption” must not beunderstood as a restriction.

SUMMARY OF THE INVENTION

The invention provides devices and automatic methods for cleaningdispensers without having to dismount the dispenser or even to removethe dispensing liquid from the dispenser capillary. The dispensersaccording to the invention do not need to comprise any moving partsother than micropumps for the cleaning process, preferably inexpensiveperistaltic micropumps. The cleaning process can be started any time,for instance periodically, or each time after the preparation of allsamples of a support plate with a certain number of sample sites, suchas 384. Frequent cleaning is essential for a robust operation of thedispenser because dispensing solutions near saturation easily formdeposits near the tip of the dispenser.

The invention can use dispensers which are known as such, where a dropof suitable size is pressed out from a capillary tip with a micropump,and is detached by any chosen process, for example by a pressure surgeof a surrounding gas flow, in such a way that it falls vertically ontothe sample site on the sample support.

The invention proposes to feed a washing fluid for dissolving thedeposit not through the inner capillary of the dispenser, but throughone or more outer channels to a location at the outer wall of the innercapillary a short distance above the tip, forming a drop completelyenclosing the tip, particularly its outside surface. As an example, oneor all of the gas channels may be used to create this drop of washingfluid around the tip of the dispensing capillary (if the means torelease the drop comprises gas channels), but it is also possible to usean extra channel (or several extra channels), which ends at or near theouter wall of the capillary (slightly) above the tip. This makes itpossible to carry out the cleaning without having to change the liquidin the inner dispensing capillary, which accelerates and simplifies thecleaning process in general.

The invention is particularly applicable for dispensers comprising anoptical device with light source and light detector to monitor andregulate the size of each individual hanging drop before it is detached.With these devices, it is not only possible to regulate the drop ofdispensing liquid, but also the drop of washing fluid. It has been foundadvantageous to form a relatively large drop of liquid solvent as thewashing fluid around the capillary tip, with a volume of about four toeight microliters, and to keep this drop in motion by, preferablyrepeatedly, drawing a part of the washing fluid into the inner capillaryand pressing it out again with the dispensing micropump, particularly toclean the interior of the tip. This motion helps to dissolve the depositbut requires monitoring the drop size to prevent the drop from fallingoff prematurely. The drop of washing fluid can subsequently be droppedinto a waste shaft. If required, this cleaning cycle can be repeatedwith new drops of washing fluid several times, before the dispensing isresumed. The washing fluid can be pumped through the gas channels or theextra channel(s) to the outer surface of the dispenser tip by a furthermicropump.

The quantities of liquid to be dispensed regularly onto the sample siteamount to only 0.5 to 1.5 microliters, and form droplets around 1.0 to1.4 millimeters in diameter. For the washing process, a larger dropvolume of about four to eight microliters has proven to be favorable,with a diameter of about 2.0 to 2.7 millimeters. In contrast to a dropof dispensing liquid which is pressed out, and then is pendant directlyand only from the tip of the central dispensing capillary, this drop ofwashing fluid should hang from a location (slightly) above the tip,enclosing completely the outside of the dispenser tip. Favorably, thedrop is attached to the exits (or outlet ends) of four or more channels,or to a small horizontal plane the material of which features a surfacetension which is substantially adhesive for the washing fluid. Evendroplets of the afore-mentioned rather large size do not detachthemselves through the effect of gravity alone when they hang from aplane with good wettability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a simple dispenser design according to the prior art, thebasic principles of which can also be used in conjunction with thepresent invention. A liquid (2) to be dispensed, such as a matrixsolution for MALDI or a mixture of matrix and analyte sample, isaspirated from a vessel (1) via a tube (3) by a micropump (4) and ispressed through the tube (5) into a drop (9). When the drop (9) is theright size, it is detached from the tip of the tube (5) by a pressuresurge of a gas (8) through the tube (7).

FIG. 2 shows the shadow cast by a spherical drop (9) in a parallel beamof light (10), for example a laser beam or other suitable collimatedbeam. The drop generates a focus (11), and the divergent light from thefocus is imaged in the detector plane (12) onto an area with a largediameter (15). If the distance between drop and detector is chosenfavorably, the shadow (13) in the original light spot (14) of theparallel beam is illuminated only by a small amount of light from thefocus, and thus a marked brightness contrast is produced between thearea of shadow (13) and the outer original light spot (14).

FIG. 3 shows the beam path of the light from a relatively large surfacearea of a light-emitting diode (20) without a drop (top) and with aspherical drop (9) (bottom). Two apertures (21) and (22) form a lightbeam, which generates a circle of full light (25) with an aperturepenumbra (26) in the plane of the detector (24). As can be seen in thelower figure, a drop (9) on the capillary (23) forms an umbra (27) withsurrounding drop penumbra (28). Here too, the light which passes throughthe drop (not shown here) is so divergent that it makes almost nocontribution to the amount of light on the detector (24). The amount oflight on the detector (24) can therefore be used to regulate the dropsize or to determine the moment at which the growing drop is detachedfrom the capillary tip.

FIG. 4a shows an embodiment of the dispenser with a dispensing micropump(46), a capillary (30) and capillary tip (31), which is surrounded by afirst cone (32) with milled gas channels (35) and a second cone (33) tocover the channels (35). A pressure surge can be used to feed a gas intothe ring duct (38) through the feed-in (34). The pressure surgecontinues through the channels (35) and detaches the drop of dispensingliquid which is not shown here. In this embodiment, the washing fluidcan be pumped in by micropump (47) through a further feed-in (36), runsthrough the channel (37) to the horizontal plane (41) above capillarytip (31), the plane (41) being wettable to hold a drop (40) of washingfluid enclosing the dispenser tip (31). It goes without saying that morethan one channel (37) can be foreseen in such a design. The size of thedrop of washing fluid is monitored photometrically and regulated by adevice comprising a light source (43) and a light detector (44), wherebythe light beam (45) casts a shadow of the drop (40) of washing fluidonto the light detector (44).

FIG. 4b depicts a similar embodiment, however without the extra channel(37). The washing fluid, for instance ethyl alcohol or some othersuitable pure liquid solvent, here is pumped in by micropump (48)through feed-in (39) into the gas channel system with at least fourchannels (35), preferably when the feed-in (34) for the gas is closed.In this case, the horizontal plane (42) is not required to be wettable,if the substantial contact of the drop (40) of washing fluid with thewashing fluid in the channels (35) is sufficient for a hold. Also inthis FIG. 4b , the drop (40) is fully enclosing the dispenser tip (31).The drop (40) solely hangs from the exits (or outlet ends) of the atleast four channels (35) and is monitored and regulated photometricallyby the device comprising light source (43) and light detector (44).After cleaning the dispenser tip by alternatingly drawing in into thecentral capillary (30) and pressing out again some of the washing fluidwith the dispensing micropump (46), a surge of gas or pumping some morewashing fluid removes the drop (40) of washing fluid, and, by reversingthe pumping direction of the micropump (48) for the washing fluid, thegas channels (35) are dried by air to remove residual solvent. The airsucked in in this way dries the inner walls of the gas channels (35) ina rather short time of about one to five minutes.

FIG. 5 depicts a typical calibration curve, as is obtained with asyringe piston pump, creating spherical drops of known volume hanging atthe tip of the dispenser. The relationship between the detector signalstrength (here in volts) and the drop volume is stable up to a volume of4 to 5 microliters and can be used for regulation of the drops ofdispensing liquid, particularly well for smaller drops up to about 3microliters. For measuring the larger drops of washing fluid up to eightor ten microliters, hanging from the horizontal plane and enclosing thetip, an independent calibration curve has to be created and used, sinceportions of the drop of washing fluid are attached around the outercircumference of the central dispensing capillary thereby being blockedfrom a field of view of the light detector (whereas a drop of dispensingliquid hanging only from the tip of the dispensing capillary is exposedto the light from the light source with virtually its entire volume).The calibration curve for drops of washing fluid should, however, have asimilar form in that the amount of light received by the detectordeclines monotonically with increasing drop size.

FIG. 6 shows how deposits on the capillary tip of the dispenser can bemeasured by virtue of the fact that, due to the shadow cast by thedeposits when no droplet of whatever kind is pendant from or at thecapillary tip, the signal strength at the detector does not return tothe original value (broken horizontal line) which it has for a cleancapillary (A). Depending on the size of the deposits (B) and (C), thereremains a smaller or larger difference from the original signalstrength. At the times (D), (F) and (H), the micropump starts to pressthe drop of dispensing liquid from the dispensing capillary; at thetimes (E), (G) and (I), the drop of dispensing liquid is detached andthe micropump may be stopped.

DETAILED DESCRIPTION

Dispensers for the microliter range usually detach a drop of dispensingliquid by mechanical, acoustic, shock wave, or hydromechanical means tomake it fall vertically onto the sample support. During dispensing ofhundreds of samples, solved material may crystallize over time asdeposit at the capillary tip of the dispenser, which impedes thevertical detachment of the drop of dispensing liquid in the medium andlong run. Therefore, frequently cleaning the capillary tip is essentialfor a robust operation of the preparation device. The invention proposesto clean the dispenser tip by creating a drop of washing fluid through achannel outside the central dispensing capillary so that it completelyencloses the dispenser tip. The size of the hanging drop of washingfluid is photometrically monitored and regulated to prevent the drop ofwashing fluid from falling off prematurely. Alternatingly drawing ininto the central dispensing capillary and pressing out again parts ofthe drop of washing fluid may help to clean the dispenser tip.

Any type of dispenser, dispensing drops in the microliter range, can beused as the basis of the invention. For example, a dispenser can beused, with which a drop of dispensing liquid is pressed out of acapillary tip and detached by any chosen process with controlledactivation so that it falls vertically onto a sample site of a samplesupport. This process can, for example, be a pressure surge in theliquid, possibly caused by a piezo element or a bubble-producing heatingelement, or a pressure surge in a gas flow surrounding the drop. Movingthe capillary upwards with a jerk can also detach the drop. The requireddroplets with a diameter of around 1.0 to 1.4 millimeters (equivalent toa volume of 0.5 to 1.5 microliters) do not detach themselves from apipette tip or a capillary by the effect of gravity alone, even if thecapillary tip is relatively thin and the force of the surface tension atthe neck of the drop is rather small.

The dispenser shown in FIG. 1 is known in principle from the prior art,but can as such also be part of a dispenser system according to theinvention. It operates as follows: the drop (9) of dispensing liquidwhich is pressed out of the inner capillary (5) by the micropump (4) isdetached, in this case without contact, by a pressure surge (8) of a gasflow from a capillary (7) surrounding concentrically the inner capillary(5).

The operation of such dispensers regularly suffers from the formation ofdeposits at the tip of the dispensing capillary when solutions nearsaturation have to be used. Material easily crystallizes at the tip andhampers correct function. Quite often a mushy mixture of microcrystalswith liquid forms a thick paste, creeping up at the outside at one sideof the tip. Then, the drops of dispensing liquid do no longer hangsymmetrically from the tip, but are drawn to one side by the deposit.Thereby the danger arises that the detached drops of dispensing liquiddo no longer fall strictly vertically, and eventually fail to hit thecorrect sample site.

Because the sometimes gel-like deposits creep up the outside of the tipduring continuing dispensing operation, the tip should be freelyprotruding from any hold by a few millimeters, so that the hold of thetip cannot be contaminated. In particular, the tip should not besurrounded by a narrow tube with a gap being formed in between (forinstance, to guide the gas surge), because a deposit within the gapbetween tip and tube is hard to remove and requires much longer cleaningprocesses.

The invention is intended for repeatedly cleaning the pipette tip of thedispenser by fast cleaning processes, either periodically or afterdetection of deposits. By software control, automatic cleaning ispossible without any help from the operator. The invention providesdevices and automatic methods for cleaning dispensers without having todismount the dispenser or even to remove the dispensing liquid from thedispenser capillary. The cleaning process can be started any time, forinstance periodically, or each time after the preparation of all samplesof a support plate with a certain number of samples, such as 384, orafter the detection of deposits at the tip of the capillary.

In contrast to time-consuming and solvent-consuming cleaning processesknown in the prior art, the cleaning according to the invention onlyneeds a short time and a tiny amount of washing fluid. The inventionproposes to feed the washing fluid for dissolving the deposit notthrough the inner capillary of the dispenser, but through one or moreouter channels to the outside of the tip of the capillary, where thedeposit is located in most cases, so that a drop of washing fluid (40)can be created which completely encloses the outside of the dispensertip (31). Favorably, the washing fluid is fed to a location a littledistance above the dispenser tip, for example amounting to about one totwo millimeters, just where the cone of the tip (31) ends. For instance,as shown in FIG. 4b , one or all of the at least four gas channels (35)may be used to create this drop (40) of washing fluid enclosing the tip(31) of the dispensing capillary. In this case, the drop (40) may hangfrom the fluid inside the exits (or outlet ends) of the at least fourgas channels (35) ending in the plane (42). On the other hand, it ispossible to use an extra channel (37), or several extra channels, whichends in a plane (41) near the tip (31) of the inner capillary, to formthe drop (40) of washing fluid, as shown in FIG. 4a . If only a singlechannel is used, as in FIG. 4a , the plane (41) is preferably configuredto be substantially wettable in order to hold the drop (40). The drop(40) of washing fluid enclosing the tip (31) of the dispenser makes itpossible to carry out the cleaning without having to change thedispensing liquid in the inner dispensing capillary (30), whichaccelerates and facilitates the cleaning process in general.

To make the drop of washing fluid fully enclose the dispenser tip, thechannels (35, 37) may end in a wettable horizontal plane (41) at whichthe drop (40) can adhere. A material providing adhesive properties forwashing fluids would be a metal, such as stainless steel or aluminum; asthe case may be, a metal layer could be applied to a suitable substrateby some evaporation process. By this means the drop (40) of washingfluid can fully enclose the dispenser tip (31) even if the outer wall ofthe tip has a surface tension which does not allow to easily wet thesurface by the washing fluid. This is the case for commercial dispensertips made from polytetrafluoroethylene (PTFE) which is unwettable bymost solvents. PTFE is the preferred material for the inner capillary(30) because it is chemically very inert.

As already mentioned, the fluid within the exits of the at least fourgas channels (35) may hold the larger washing drop (40) even if theplane (42) is not particularly wettable. However, also the plane (42)from FIG. 4b could comprise an easily wettable material.

The washing fluid can be pumped to the dispenser tip (31) by a furthermicropump (47 or 48) to form the drop (40). Preferably a pure liquidsolvent, e.g. ethyl alcohol, may serve as washing fluid, capable tosolve the crystalline deposit. It has been found advantageous to form arelatively large drop of solvent on the capillary tip (31), with avolume of around 4 to 8 microliters, controlled and regulated by anoptical drop size measuring unit comprising a light source (43) and alight detector (44). After forming the drop (40) of washing fluidthrough the outer channel(s) (35 or 37), it is kept motionless for about30 seconds to soak the deposits at the outside of the tip. Subsequently,the drop (40) of washing fluid is brought in motion for another 30seconds by drawing a part of the washing fluid from the drop into thecapillary tip (31) and pressing it out again with the dispensingmicropump (46), again photometrically monitored and controlled. Asdispensing micropump (46), either a motorized syringe piston pump may beused or, much less expensive, a small peristaltic pump, in which a tubeis pressed together in a bed by moving rollers. This motion of the drop(40) of washing fluid dissolves the deposits, particularly also in theinner part of the tip (31). The drop (40) of washing fluid can then bedropped into a waste shaft (not shown) by a pressure surge or by anincrease of the drop size, that is, more washing fluid is suppliedthrough the outer channel(s) (35, 37) until the detachment threshold dueto gravity is exceeded. Subsequently, a new drop (40) of washing fluidmay be formed in order that the washing cycle is repeated with cleanwashing fluid. Finally, the drop (40) of washing fluid may be removed bya gas surge. By changing the pumping direction of the washing fluidmicropump (48), first the residual washing fluid and then air is drawnin to dry the gas channels (35), needing about one to five minutes forexample. A full cleaning process may consist of three to ten washingcycles, each with a new drop (40) of washing fluid. If required, thiswashing process can be repeated several times, but in general it hasbeen found that the process is very efficient so that a single cleaningprocess may be sufficient.

Even if three to ten drops (40) are used in subsequent cycles of thecleaning process to clean the tip (31) of the capillary, less than 100to 200 microliters washing fluid is consumed in a single cleaningprocess. With a bottle of about 200 milliliters of washing fluid, 1000to 2000 cleaning processes can be performed, each taking a few minutesonly.

The generation of the rather large drop (40) of washing fluid and themotion of the drop by drawing in and pressing out some of the drop fluidendangers the hold of the drop; the drop (40) of washing fluid may falloff prematurely, particularly if inexpensive peristaltic pumps are usedwhich do not work smoothly. Therefore, the invention is particularlyapplicable for contactless dispensers comprising an optical device withlight source (43) and light detector (44) to monitor and regulate thesize of each individual hanging drop (40) before it is detached, asshown schematically in FIG. 3. With these devices, it is not onlypossible to monitor and regulate the drop (9) of dispensing liquid (FIG.3), but also the drop (40) of washing fluid (FIGS. 4a and 4b ), using anextra calibration curve which, however, will bear some resemblance tothe calibration curve of the drop of dispensing liquid as it will show amonotonic decline with increasing size of the drop.

The quantities of liquid to be dispensed onto the sample sites regularlyamount to only 0.5 to 1.5 microliters, and form droplets around 1.0 to1.4 millimeters in diameter. For the washing process, a drop volume of 4to 8 microliters has proven to be favorable, with a diameter of about2.0 to 2.7 millimeters, enclosing the dispenser tip. Droplets of thissize are in danger of falling off prematurely by irregularities of thepumping speed of peristaltic pumps or other irregularities. Increasingthe drop of washing fluid to about 10 to 20 microliters lets the dropfall, due to the gravity force, wherein the precise volume is dependingon the surface tension at the drop's neck.

For monitoring and regulating the size of the drops, a photometricdevice may be used, as schematically shown in FIGS. 4a and 4b . Althoughthe drops are usually quite transparent, the light beam from a lightsource, which is guided past the capillary tip of the dispenser, casts aclearly visible shadow of the generally round drop onto the detectorbecause the light incident on the drop is strongly focused by the largedifference in the refractive index between air and liquid (see FIG. 2).The light falling onto the drop is so divergent beyond the near focalpoint that it contributes almost nothing to the amount of light at thelocation of the detector, given suitable spacing between light source,drop and detector. The shadow reduces the amount of light on thedetector and the resulting signal strength can thus be used to regulatethe drop size for dispensing or washing. It is preferable if thediameter of the detector is greater than the shadow cast by the largestdrop, whose size is still to be regulated.

When evaluating the amount of light on the detector in the presence of adrop at or around the dispenser tip, the rather irregular dimensions ofa drop of washing fluid, pendant from a point slightly set back from thetip of the dispensing capillary, compared to the rather regularspherical shape of a drop of dispensing liquid, pendant directly andonly from the quite sharp capillary tip, will have to be accounted for.For instance, as indicated in FIGS. 4a and 4b the drop (40) of washingfluid is not strictly spherical but has a round convex portion extendingbeyond the tip of the dispensing capillary (31) and actually beingexposed to the light beam (45) from a light source (43). The opticalimaging properties of this convex portion, such as the focal length,differ quantitatively but not necessarily qualitatively from those of aspherical drop used for the explanation of the basic principles ofphotometric monitoring and regulation that follows.

As can be seen in FIG. 2, a spherical drop (9) forms a clearly visibleshadow in a parallel beam (10), although from a strictly physical pointof view, a shadow is only defined for a body made from a non-transparentmedium. It would be scientifically more correct to talk about adifference in contrast, but the term “shadow” will be used continuouslyin the following for the sake of clarity. The shadow forms as a resultof the optical deflection of the light which falls onto the drop, due tothe refractive power of the drop. The light incident on the drop isfocused with a short focal length by the lens effect of the drop. Beyondthe near focal point (11), the light is so divergent that it hardlylightens the cast shadow (13) if the distance between the drop (9) andthe detector (12) is suitably chosen. If the detector area (not shownhere) in the detector plane is only slightly larger than the shadow castby the largest drop to be regulated, for example with a detectordiameter as large as the diameter (14) of the light beam, the amount oflight which falls onto the detector can be used to monitor and regulatethe drop size and, in particular, to determine the moment at which thedrop is about to be detached from the dispenser tip.

The relationship between the light signal of the detector and the volumeof the drop must be determined by calibrations. FIG. 5 presents acalibration curve for spherical drops freely hanging on the dispensertip. The calibration can be carried out very easily with an accuratesyringe piston pump. Up to a volume of four to five microliters, astable and reproducible signal is obtained, which is very well suitedfor the regulation. For drops of washing fluid, for instance hangingfrom a substantially wettable plane, fully enclosing the dispenser tipand thereby having a rather irregular shape, a separate calibration hasto be performed that accounts for the fact that a focal length, or inother words the light transmission properties of the drop, cannot be aseasily defined as in the case of a spherical drop. It has been found,however, that, once a proper calibration is established, both types ofcalibration can be transferred within the required accuracy toregulating systems of the same design.

Monitoring and regulating the size of a drop (40) of washing fluid maybe performed as follows: once a cleaning cycle is initiated, an amountof light emitted from the light source (43) and received by the lightdetector (44), which in favorable embodiments can be located at aposition diametrically opposite to the position of the light source (43)in relation to a dispenser axis (not shown), is evaluated as to whichamount of washing fluid is currently attached at and around the tip (31)of the dispensing capillary. If the detected amount is not sufficientfor the drop of washing fluid fully enclosing the capillary tip, acommand signal is issued to the micropump (47, 48) for supplying thewashing fluid that more washing fluid has to be delivered. The micropump(47, 48) will then pump more washing fluid through the outer channel(s)(35, 37) to the capillary tip (31) and, after a predetermined period ofpump operation, a further light detector reading will be made. If aminimum threshold of drop size, which indicates that the size of thedrop of washing fluid is sufficient for enclosing the capillary tip inits entirety, is exceeded the micropump will receive a command signal tostop operation and the drop of washing fluid will be kept hanging at andaround the capillary tip for a predetermined period of time, such asabout several seconds up to minutes, so that deposit dissolving can takeeffect. Then, the drop of washing fluid can be disposed into waste,which requires that the dispenser device and a waste shaft have beenbrought into alignment beforehand.

Optionally, after a first period of “passive” dissolving in which thedrop (40) of washing fluid just hangs at and around the capillary tip(31), in a second period of “active” dissolving, parts of the drop (40)of washing fluid may, alternatingly, be drawn into the capillary tip andpressed out again, as the case may be repeatedly, by correspondinglyback and forth operating the dispensing micropump (46) for the centralcapillary (30). In particular during this second period of activedissolving, further light detector readings are made to avoid that thesize of the drop of washing fluid exceeds a maximum threshold of dropsize, which indicates that the size of the drop (40) of washing fluidapproaches the critical size at which detachment due to gravity pullwould occur, in a phase of pressing out. If the maximum threshold isreached, the dispensing micropump (46) receives a command signal toeither start a period of reverse operation to draw in the drop ofwashing fluid into the capillary tip again or to stop further operationand, if a predetermined period of active dissolving, such as betweenseveral seconds up to minutes, has finished, the washing micropump (47,48) receives a command signal to start an operation of disposing thedrop of washing fluid into waste, as the case may be jointly with themicropump (46) for the dispensing liquid.

Due to the irregular dimensions of the drops of washing fluid (ratherpear-shaped than spherical), the aforementioned minimum and maximumthresholds are preferably determined empirically and may correspond tocertain voltage values, as shown in FIG. 5 on the y axis, if aphotoelectric cell is used as light detector, for instance.

The light source and the light detector are preferably arranged andaligned such that the tip of the capillary slightly projects into thelight beam. It is then possible, with this device, to recognize depositson the capillary tip of the dispenser, for example by monitoring a basesignal of light which is present when no liquid is being pressed out ofthe capillary tip (and even the meniscus is retracted), or by monitoringstray light with a second photoelectric cell or other suitable lightdetector. The second photoelectric cell or light detector is preferablylocated at a position off the axis of the light beam from the lightsource. Deposits on the capillary tip of the dispenser caused bycrystallizing material can be detected in this way. Detection of adeposit can trigger an automatic cleaning of the dispenser capillary, insome cases even during a deposition sequence to ensure the reliablespotting of drops of dispensing liquid.

In an embodiment with a parallel light beam according to FIG. 2, theshadow of the drop, which has no penumbra here, can be produced using alight beam from a laser diode or a small laser. Since the beams fromrelatively low-cost laser diodes or small lasers have only smalldiameters, the laser beam must be expanded by a telescope-like lenssystem to a diameter which is larger than the drop.

In the embodiment according to FIG. 3, an optical device with anextended light source (20) and a light detector (24) can be seen. Alight beam from the surface area of a light source (24), limited by theapertures (21) and (22), casts an umbra (27) of the spherical drop withsurrounding penumbra (28) of the drop onto the detector; here also, itis possible to use the residual light on the detector to regulate thedrop size. Here too, the light, which is focused by the spherical formof the drop (9) (not shown in FIG. 3 for reasons of clarity), becomes sodivergent beyond the focal point that it makes hardly any contributionto the amount of light on the detector (24) if the spacings at thelocation of the detector are chosen appropriately.

The light beam can be generated by laser diodes, but preferably bysimple and more economic light-emitting diodes (LEDs) of sufficientluminosity, and can be formed or restricted by lenses, but preferably bysimple apertures, in such a way that a sufficiently large umbra of thedrop is generated. The light detector can preferably be a photoelectriccell or charge-coupled device (CCD). The arrangement of FIG. 3 depictsthe forming of the light beam by simple apertures.

For a preferred embodiment according to FIG. 3, a simple and verylow-cost light-emitting diode (LED) of sufficient size and luminance canbe used as the light source (20), with a power supply which is alsolow-cost. The light beam of a sufficiently bright light-emitting diode(20) is simply restricted by two apertures (21) and (22) here in orderto provide proper collimation, and forms a low-cost system that issimple and stable with regard to accurate positioning, and generates anumbra (27) and a surrounding penumbra (28) from the drop.

As has been noted above, it is preferable here to detach the drop ofdispensing liquid by a pressure surge of a gas blowing against it. Itis, however, not easy to keep two capillaries (5) and (8), the inner onefor forming the drop of dispensing liquid, the outer one for thedetaching gas surge, exactly coaxial, as shown in FIG. 1. Even a slightdeviation may have the effect that the drop (9) is no longer detachedvertically. For this reason, devices as shown in FIGS. 4a and 4b arepreferred. Here, a cone (32) which closely surrounds the dropletcapillary (30) has at least two, preferably four or more, outer channels(35) covered by the cone (33), which direct the gas surge towards a dropof dispensing liquid (which is not the drop (40) of washing fluid shownin these figures) at the tip (31) of the dispensing capillary (30) andforce it to fall off by abruptly exerting a gas pulse against it. Thepressure surge can be generated by a tiny piston pump, for example, andfed to the admission aperture (34). The piston pump can consist of asimple piston in a cylinder, where the piston is moved by a simpleelectromagnet and reset by a spring so that the cylinder can be filledand emptied. The resetting by the spring could then generate thepressure surge.

If deposits have to be removed, the dispenser capillary can be cleanedautomatically. For each washing cycle of the simple cleaning process, asecond micropump (47, 48), preferentially a low-cost peristaltic pump,feeds the solvent as the washing fluid for dissolving the deposit, as isshown in FIG. 4a , not through the inner capillary (30), but through anouter channel, for example through an additional milled channel (37) inthe above-mentioned cone (32), which closely surrounds the innercapillary (30). Instead, one or all of the gas channels (35) may be usedto feed the washing fluid to the tip (31) of the dispensing capillary,as shown in FIG. 4b . If the tip protrudes from a flat horizontal plane(41) with good wettability, such as by about one or two millimeters, thedrop (40) of washing fluid will hang down from this flat area and fullyenclose the dispenser tip. The size of the drop (40) of washing fluid ismonitored by the photometric device (43, 44). This external feedingprocess makes it possible to carry out the cleaning without having tochange the dispensing liquid in the inner capillary (30), whichaccelerates the process in general. The cleaning can be supported by amotion of the drop (40) of washing fluid, caused by alternatinglydrawing into the inner capillary (30) and pressing out again some of thedrop (40) of washing fluid by the dispensing micropump (46). Also inthis period the drop of washing fluid is monitored and controlled by thephotometric device in order that, during the pressing-out phase, thedrop (40) of washing fluid does not grow too large as a consequence ofwhich it would be unintentionally falling off from the tip (31)prematurely due to the gravity pull. After a washing cycle, the drop(40) of washing fluid is detached by pumping some more washing fluidinto it. At the end of the cleaning process, after a predeterminednumber of washing cycles, such as three to ten, the washing fluid can beremoved by a gas surge through the gas channels, and the washingchannels can be dried by fast sucking air by the washing micropump (48)for about one to five minutes.

The cleaning systems can be beneficially used to switch the wholedispenser system into a standby state when sample support plates have tobe exchanged, or when the whole system should be switched down for ashort period. An amount of liquid corresponding to one or more drops ofwashing fluid may be sucked into the dispensing capillary, either indirect contact with the dispensing fluid, or even with an air bubble inbetween by first sucking a bit of air into the dispensing capillary.This measure of closing the capillary with some washing fluid serves tokeep the tip of the dispenser from being contaminated by crystallizingmaterial. The standby state can thus be kept for hours or even dayswithout having to empty the dispenser capillary. After switching onagain, the washing fluid and some of the dispensing fluid is drippedinto waste, to bring fresh and undiluted (and unpolluted) dispensingliquid to the tip.

This cleaning system has several advantages: cleaning can be performedautomatically without manual intervention; the cleaning process ishighly timesaving; a stand-by mode without the danger of crystallizationis possible; furthermore, the consumption of washing fluid is very low(a bottle with 200 milliliters ethyl alcohol may serve for 1000 to 2000cleaning processes, for instance).

Several dispenser systems, either with individual monitoring andregulating device for the drop size, or with a common photometricmeasuring device for all dispensers, can be used to prepare the sampleswith several different dispensing liquids. Dispenser systems withcleaning devices according to the principles of the invention areparticularly advantageous when working with dispensing solutions,particularly with solutions near saturation which are particularly proneto crystallization of deposits.

The invention has been shown and described with reference to a number ofdifferent embodiments thereof. It will be understood, however, thatvarious aspects or details of the invention may be changed, or variousaspects or details of different embodiments may be arbitrarily combinedif practicable, without departing from the scope of the invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limiting the invention,which is defined solely by the appended claims.

1. A method for cleaning a tip of a dispensing capillary, wherein awashing fluid is fed through at least one outer channel to a locationslightly set back from the tip of the central dispensing capillary toform a drop fully enclosing the tip, wherein the size of the drop ismonitored and regulated photometrically by a device having a lightsource and a light detector to prevent the drop of washing fluid fromfalling off prematurely.
 2. The method according to claim 1, wherein thedrop of washing fluid is formed at a horizontal plane beingsubstantially wettable by the washing fluid so that the drop can fullyenclose the tip even if a tip material is substantially unwettable bythe washing fluid.
 3. The method according to claim 1, wherein the dropof washing fluid is kept in motion by at least once drawing it partlyinto the dispensing capillary and pressing it out again with the aid ofa dispensing micropump used to dispense liquids through the dispensingcapillary.
 4. The method according to claim 1, wherein an amount oflight received by the light detector in a phase where no drop is pendantfrom an end of the at least one outer channel is used to detect depositsat the tip of the dispensing capillary.
 5. The method according to claim1, wherein some washing fluid is drawn into the dispensing capillary toprotect the tip from being contaminated with crystallizing materialduring standby times.
 6. The method according to claim 1, wherein, incertain phases of operation, a first micropump presses a drop ofdispensing liquid out of the tip of the dispensing capillary and adetachment device detaches the drop of dispensing liquid from the tip ofthe dispensing capillary, and wherein, in alternate phases of operation,a second micropump presses the washing fluid through the at least oneouter channel outside of the dispensing capillary to form the drop ofwashing fluid hanging from the end of the at least one outer channel andfully enclosing the dispensing capillary tip.
 7. The method according toclaim 6, wherein the detachment device for dispensing a drop from thetip of the dispensing capillary comprises one of a device to generate apressure surge of a gas, a device for jerky detachment, a piezo device,and a vapor-bubble generator.
 8. The method according to claim 6,wherein at least one of the first micropump and the second micropump isa peristaltic micropump or a piston pump.
 9. The method according toclaim 1, wherein the at least one outer channel ends in a flathorizontal plane a material of which is configured such that it hassubstantial adhesive properties for being wettable by the washing fluid,in order to enable the drop of washing fluid to fully enclose thedispenser tip even if a material of the dispenser tip is substantiallyunwettable by the washing fluid.
 10. The method according to claim 8,wherein the material of the flat horizontal plane comprises metal. 11.The method according to claim 1, wherein the material of the dispensingcapillary comprises polytetrafluoroethylene (PTFE).
 12. The methodaccording to claim 1, wherein the tip of the dispensing capillaryprojects into the light beam between light source and light detector tophotometrically detect deposits when no drop is pendant from thecapillary tip.
 13. The method according to claim 1, wherein the lightsource and the light detector are located diametrically across adispensing capillary axis.
 14. The method according to claim 1, whereinthe light source is one of a light-emitting diode (LED), a laser and alaser diode and comprises lenses or apertures as light-beam formingelements.
 15. The method according to claim 1, wherein the lightdetector is one of a photoelectric cell and a charge-coupled device(CCD).
 16. The method according to claim 1, wherein at least one of thefirst micropump and second micropump perform an oscillatory backward andforward pumping of liquid.
 17. The method according to claim 1, whereinthe at least one outer channel comprises one of an extra channel forsupplying washing fluid and a gas channel for exerting gas pulses ondrops hanging from, or at, the tip of the dispensing capillary.
 18. Themethod according to claim 1, wherein the at least one outer channel forsupplying washing fluid comprises two or more channels being arranged ina rotationally symmetric manner around the dispensing capillary.
 19. Themethod according to claim 1, wherein a volume of the drop of washingfluid amounts to between about four and eight microliters.